PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDA) has the highest 1-, 5-, and 10- year mortality because more than 80% of patients present with locally advanced or metastatic disease, precluding an attempt at surgical resection. PDA is characterized by a robust desmoplastic reaction that promotes cancer cell dissemination through direct interactions with tumor epithelial cells (TECs) and surrounding stromal cells, including cancer- associcated fibroblasts (CAFs). This desmoplasia also makes PDA unusually resistant to systemic chemotherapies by acting as a physical barrier sheltering it from treatment. Our lab previously identified hyaluronan (HA) as a defining feature of this desmoplasia that generates remarkably elevated interstitial pressures capable of collapsing intratumoral vasculature. HA is a unique hydrophilic glycosaminoglycan composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. It is synthesized by the HA synthase (Has) family of enzymes, Has1, Has2, and Has3, of which Has2 produces the highest molecular weight (MW) polymers. This is important because higher MW HA polymers bind greater amounts of water and expand more than lower MW species, resulting in greater swelling pressures. In PDA epithelial cells, we found that HA was almost exclusively produced by Has2. In addition to its mechanical contributions to the PDA microenvironment, HA also functions as a signaling molecule to promote cancer progression. HA forms a pericellular coat around tumor cells through interactions with HA-binding proteins at the cell surface, which assists in tumor cell extravasation and anoikis resistance during metastasis. HA also binds cell surface receptors, including CD44, RHAMM, and ICAM-1, to induce signaling in TECs and CAFs. HA signaling in TECs and CAFs promotes tumor initiation, progression, invasion, angiogenesis, metastasis, and drug resistance. Using a genetically engineered mouse model of pancreas cancer, KrasG12D/+;Trp53R172H/+;p48Cre/+ (KPC), that faithfully recapitulates the pathobiology of the human disease, we demonstrated that enzymatic degradation of intratumoral HA with pegylated hyaluronidase (PEGPH20) drastically reduced interstitial gel- fluid pressures and improved perfusion and response to chemotherapy. This combined enzymatic and chemotherapy strategy has progressed rapidly in the clinic and is now in a global Phase 3 trial. Recently, we developed models to conditionally delete Has2 heterozygously (KPHC) and homozygously (KPHHC) in the pancreatic epithelial cells of KPC mice. We have observed a striking reduction in metastatic burden and alteration of HA-receptor expression and signaling in KPHC and KPHHC PDA compared to KPC. I propose to investigate the autocrine and paracrine mechanism(s) of HA biology that drive PDA metastasis. The goals of these investigations are to understand the pathogenic implications of tumor epithelial cell-HA production in autocrine signaling, paracrine signaling to CAFs, and systemic circulation that combine to promote metastasis.